This invention relates to chromosome identification and, more particularly, to identifying repetitive DNA sequences which hybridize to specific chromosomes.
As is well known, living cells include chromosomes, those structures which carry DNA genetic units. Specific sets of chromosomes determine the characteristics of the life associated with the DNA genetic units. Thus, human cells contain 24 different chromosome types (22 autosomes, X and Y) and the number and arrangement of chromosomes within the cell are indicative of various normal and abnormal developments. By way of example, normal cells contain a pair of each type autosome and the presence of three of a chromosome type (trisomy) may be indicative of an abnormal condition. Trisomy 9 (i.e., three number 9 chromosomes) is indicative of a syndrome which is fatal within a few days of birth. Trisomy 16 is present in one-third of all spontaneous abortions that contain chromosome trisomies. Trisomy 21 is indicative of Down's syndrome.
Repetitive DNA clones have been described which hybridize to groups of human chromosomes, often with a prominent site of hybridization on only one or two autosomes. See, e.g., A. R. Mitchell et al., "Molecular hybridization to meiotic chromosomes in man reveals sequence arrangement on the No. 9 chromosome and provides clues to the nature of `parameres`," Cytogenet. Cell Genet. 41: 89-95 (1986), which teaches a probe, Xbl, which hybridizes preferentially, but not exclusively, with chromosome 9.
It has been suggested that each human chromosome may be characterized by one or more distinct subsets of alpha satellite DNA. See, e.g., P. Devilee et al., "Two Subsets of Human Alphoid Repetitive DNA Show Distinct Preferential Localization in the Pericentric Regions of Chromosomes 13, 18, and 21," Cytogenet. Cell Genet. 41, 193-201 (1986); A. Lund Jorgensen et al., "Chromosome-Specific Subfamilies Within Human Alphoid Repetitive DNA," J. Mol. Biol. 187, 185-196 (1986). Indeed, the enrichment of one or a few distinct subfamilies on individual chromosomes is reported. However, no process is taught for systematically determining nucleotide sequences which form chromosome specific probes. Further, no probes are taught which are chromosome specific under a normal stringency requirement for a positive chromosome identification after in situ hybridization of the probe with chromosomes within a cell.
It will be appreciated that the chromosome specificity of chromosome preferential DNA probes identified in the prior art may be improved by increasing the stringency under which hybridization is attempted. However, hybridization under conditions of high stringency may not obtain reproducible results. While only specific chromosomes may be tagged, not every such chromosome may be tagged, a clearly unacceptable condition for a routine clinical application. Further, high stringency levels frequently reduce the size and/or intensity of the hybridization site, requiring increased amplification of the fluorescent signal or longer time of autoradiographic exposure. These conditions increase the nonspecific background errors from the measurements. Probes that are chromosome specific after hybridization at normal stringency and a single amplification step or short exposure would be more useful for most applications, especially for identification and quantitation of chromosomes in interphase nuclei.
The ability to tag specific human autosomal chromosomes in interphase nuclei would allow investigations of the three-dimensional structure of the nucleus to be conducted and provide an approach to chromosome analysis where the isolation of metaphase chromosomes is inconvenient, difficult, or impossible. However, any cross hybridization between a chromosome "specific" probe sequence and other chromosomes dilutes the usefulness of the probe, especially for hybridization to interphase nuclei. The present invention provides a process for identifying repetitive nucleotide sequences which hybridize in situ to specific chromosomes under normal stringency conditions to enable such investigations and analysis to be reliably conducted.
Accordingly, it is an object of the present invention to identify repetitive nucleotide sequences which are chromosome specific under conditions of normal stringency.
It is another object of the present invention to enable in situ identification of chromosomes in intact cellular nuclei.
Still another object of the present invention is to provide a method for determining probe sequences which contain only that portion of a repeat unit that is enriched on or unique to the chromosome being identified.
One other object of the present invention is to identify chromosome specific nucleotide sequences having a length that can be synthesized.